Purification and properties of phenylalanyl aminopeptidase synthesised by Pseudomonas sp.

Intracellular aminopeptidase synthesized by a soil strain of Pseudomonas sp. was purified 323-fold using the following procedure: saturation with ammonium sulfate, separation by preparative electrophoresis, anion-exchange chromatography and gel filtration chromatography. Molecular weight of the enzyme determined according to the latter method was 57 kDa. Aminopeptidase showed a high substrate specificity and affinity to Phe-beta-naphtylamide (Phe-beta-NA) as a substrate. A considerable inhibition of the enzymatic activity by iodoacetamide and p-chloromercuribenzoate (p-CMB) led to the conclusion that it was a cysteine aminopeptidase. Hydrosulphide compounds markedly stabilised the enzyme. Ethylenediaminetetra-acetic acid (EDTA), a metalloenzyme inhibitor, caused a double increase in the phenylalanyl aminopeptidase activity. Mg2+ ions activated the enzyme to a negligible extent, whereas Co2+, Cu2+, Cd2+ and Pb2+ ions contributed to its inhibition. The highest enzymatic activity was observed at 37 degreesC and pH 7.0.